1. Nonmuscle Myosin II Exerts Tension but does not Translocate Actin-Filaments in Vertebrate Cytokinesis In vertebrate cells, nonmuscle myosin II (NM II) and cytoplasmic actin compose two major contractile proteins at the contractile ring mediating cytokinesis. It is generally thought that contractile ring constriction is driven by NM II translocation of antiparallel actin-filaments, similar to the sliding filament model in muscle contraction. Using a combination of in situ (COS-7 cells treated with siRNA and blebbistatin), kinetic (stopped flow experiments with baculovirus-expressed protein), as well as in vivo (genetically altered cardiomyocytes in mouse hearts) approaches we provide evidence that NM II translocation of actin is not essential for vertebrate cytokinesis. The three NM II paralogs (II-A, II-B and II-C), similar to skeletal muscle myosin, are composed of catalytically active heads, which bind to and translocate actin in an ATP-dependent manner, and are also composed of bipolar filament-forming rods. NM II-B is the major paralog expressed in cardiac myocytes and COS-7 cells. Ablation of NM II-B in mouse cardiac myocytes in vivo or siRNA depletion of NM II-B in cultured COS-7 cells results in cytokinesis defects indicating the requirement of NM II-B for cytokinesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits the rate of ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not the rate-limiting factor for cytokinesis. Blebbistatin inhibition of cytokinesis further reveals the importance of myosin strongly binding to actin thereby exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the mechanochemical properties of mutant NM II support efficient tension maintenance despite loss of the ability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of essentially all NM II heads to actin, thereby increasing tension and resisting expansion of the ring and cell cortex. Importantly, in the three dimensional context of the mouse heart in vivo, mutant NM II-B R709C that cannot translocate actin filaments also rescues multinucleation in NM II-ablated cardiac myocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and crosslink actin-filaments and to exert tension on actin during contractile ring constriction. 2. Nonmuscle Myosin II-B Is Required for Epicardial EMT in Coronary Vessel Formation Mice ablated for nonmuscle myosin II-B (NM II-B) die by E14.5 due to heart defects. To better understand the role of NM II-B in late heart development we used transgenic mice expressing Cre-recombinase driven by the SM22 promoter to conditionally ablate NM II-B in cardiac myocytes and epicardial cells but not in endocardial and fibroblast cells. BSM22 /BSM22mice developed arrhythmias as early as 1 monthof age and died suddenly by 6 months in dilated right ventricular failure. Analysis of BSM22 /BSM22 hearts shows significant defects in coronary vessel formation especially in the right ventricle with progressive loss of right ventricular cardiac myocytes. BSM22 /BSM22 hearts are also hypoplastic with defects in cytokinesis in the cardiac myocytes. To further study the cellular mechanisms of coronary vessel defects in BSM22 /BSM22 hearts, we ablated NM II-B specifically in epicardial (BWT-1/BWT-1 mice) or myocardial (BNkx/BNkx mice) cells using WT-1 and Nkx2.5 Cre mice, respectively. Both BWT-1/BWT-1 and BNkx/BNkx mice died during embryonic development with hypoplastic hearts. Interestingly, while the BWT-1/BWT-1 hearts showed severe defects in coronary vessel formation, BNkx/BNkx hearts showed marked defects in cardiac myocyte cytokinesis. Consistent with abnormal coronary vessel formation in BWT-1/BWT-1 hearts, studies employing epicardial explants in collagen gels show that NM II-B ablated epicardial cells develop defects in branching morphogenesis. Similar defects were observed in wild type epicardial explants when treated with blebbistatin which inhibits NM II enzymatic activity. In addition epicardial explants prepared from mice expressing motor impaired NM II-B also show branching abnormalities, indicating a requirement for NM II-B enzymatic (motor) activity. Although NM II-A is also expressed in epicardial cells, ablation of NM II-A does not affect coronary vessel formation. Our results provide evidence for a novel role for NM II-B in coronary vessel formation during mouse heart development.